Bioreactor apparatus

ABSTRACT

A bioreactor apparatus for producing metabolites from bioorganisms by depositing the animal cells on a carrier made of a porous material. The apparatus includes a hermetically sealed container (1) having a first supply channel A, for supplying a liquid containing animal cells into the inside thereof, a second supply channel (B) for supplying a culture solution and a substrate solution into the inside thereof, a first exhaust channel (a) for exhausting a liquid containing suspended animal cells from the inside thereof, and a second exhaust channel (b) for exhausting a residual part of the culture solution, a residual part of the substrate solution and a solution containing metabolites of the animal cells from the inside thereof. Disposed within the container is an animal culture base including a porous carrier (31, 33, 34) and a culture bed (4) arranged so that liquid supplied from the supply channels (A, B) are brought into contact therewith while flowing in the inside of the container (1). Further, the apparatus includes a filtration membrane (6) arranged across the second exhaust channel (b) in the inside of the container (1) and provided for filtrating a liquid to be exhausted through the second exhaust channel (b).

This is a continuation of application Ser. No. 07/720,048 filed Jun. 24,1991, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to a bioreactor apparatus, andparticularly relates to a bioreactor apparatus for producing metabolitessuch as human body cure vaccines, interferons, cancer antigens,hormones, cell growth factors, lymphocines, various kinds of catalysts,and the like, from bioorganisms such as various kinds of animal cellsand the like through depositing the animal cells on a carrier made of aporous material such as nonwoven fabric or the like, and cultivating theanimal cells with a high yield and density on the carrier, by whichvarious useful physiologically active substances can be producedselectively, continuously and efficiently.

For in-vitro production of useful substances produced by various humancells, such as human immuno chemical mediators, human type enzymes,etc., for medicinal application it, is necessary to establish both atechnique for mass-cultivating various kinds of animal cells and developa high-performance bioreactor apparatus.

The animal cell cultivating techniques used heretofore include "asuspension growth technique" for growing cells suspended in an aqueoussolution, "a microcarrier growth technique" for growing suspended cellsby adhering the cells with porous microbeads having a diameter of about200 μm, "an adhesion carrier growth technique" for growing cells bycarrying the cells on an adequate nonporous and/or porous carrier (suchas porous film, hollow fiber, nonwoven fabrics etc), and the like.

Japanese Patent Unexamined Publication No. Sho. 59-59187 discloses acultivating apparatus for performing the adhesion carrier growthtechnique in which animal cells are adhered or grown on various forms ofculture supports housed in a culture container of the apparatus while aculture solution is sprayed and supplied into the container.

Japanese Patent Unexamined Publication No. Sho. 56-42584 discloses acultivating apparatus designed to pack hollow fibers in a culturecontainer fill space between a wall of the container and an outersurface of the hollow fiber with suspended cells and supply a culturesolution from the inside of the hollow fiber. Further, Japanese PatentUnexamined Publication No. Sho. 64-34276 discloses a material forperforming the adhesion carrier growth technique, in which nonwovenfabric made from specific superfine fiber is used as a carrier for acell culture bed and is arranged in a plastic schale (laboratory dish)to cultivate animal cells.

However, in the conventional suspension growth technique and themicrocarrier growth technique, the concentration of floated or suspendedanimal cells is limited to a maximum of about 10 g/l. Further, theconcentration of the substrate solution cannot be increased because ofsubstrate inhibition and osmotic pressure. Furthermore, shear forcesimposed on cells in the solution by contact, collision, abrasion, etc.,of the cells with the fluidal change of solvent are so large that thecells are often inactivated or killed. In the microcarrier growthtechnique, the cells are, in most cases, separated or dropped from themicrobeads. In the adhesion carrier growth technique, it is not onlydifficult to continuously circulate and exchange the culture solutionand the substrate solution and to supply the necessary oxygen forcultivating animal cells, but the contact of the cells with the culturesolution and the substrate solution is uneven, insufficient andinefficient. Furthermore, metabolites cannot be continuously separatedto the outside of the apparatus. As a result, nutritional balancedisorder caused by excess or shortage of the substrate and cultureenvironment disorder caused by storage of the metabolites occur locallyin the cells. Consequently, animal cells cannot be cultivated in a highyield and a high density per volume. Further, efficient metaboliteproduction is impossible. Among the conventional apparatuses forperforming the aforementioned techniques, there is no apparatus forsolving these problems.

SUMMARY OF THE INVENTION

The present inventors have made intense studies to solve theaforementioned problems in the prior art in order to arrive at thepresent invention. Accordingly, the present invention is directed to abioreactor apparatus comprising: a hermetically sealed container havinga supply channel A for supplying a liquid containing animal cells intothe inside thereof, a supply channel B for supplying a culture solutionand a substrate solution into the inside thereof, an exhaust channel afor exhausting a liquid containing suspended animal cells from theinside thereof, and an exhaust channel b for exhausting a residual partof the culture solution, a residual part of the substrate solution and asolution containing metabolites of the animal cells from the insidethereof; an animal cell culture base composed of a porous carrier and aculture bed provided on the carrier and arranged so that liquidssupplied from the supply channels are brought into contact with eachother while flowing in the inside of the container and then led to theexhaust channels; and a microfiltration membrane arranged across theexhaust channel b in the inside of the container for filtrating a liquidto be exhausted to the exhaust channel b. In particular, the porouscarrier is made of nonwoven fabric. Further, the animal cell culturebase is arranged so as to be entirely penetrated by the substratesolution supplied into the container. In the case where the base isshaped like a film, the base is arranged. spirally or arranged in theform of a laminate composed of a large number of sheets. In particular,the animal cell culture base is adapted for adhesion and fixation ofanimal cells to be cultivated. The liquid supplied into the containercontains nutriments necessary for cultivating animal cells or contains asubstrate for acting on cultivated animal cells to produce apredetermined metabolite. The liquid supplied into the containercontains oxygen in a necessary and sufficient amount for the dualpurpose of cultivating animal cells and maintaining activity thereof. Inparticular, the liquid exhausted from the container contains at leastone substance produced by cultivated animal cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a sectional view of a bioreactor apparatus filled withlaminate-type culture film as an embodiment of the present invention;and

FIG. 2 is a sectional view of a bioreactor apparatus filled withspiral-type culture film as another embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described below in detail.

The hermetically sealed container used in the present invention is ashell member of a bioreactor apparatus and a container for cultivatedanimal cells to produce a target useful substance from the cultivatedanimal cells. Examples of preferred materials for the container arestainless steel, ceramics such as ceramics, hard glass, etc.,heat-resisting polymer capable of being treated with hot steam such aspolycarbonate, polysulfone, polyethersulfone, polyetheretherketone,etc., and the like. In particular, in the case where observation of theinside of the container is required, it is preferable that the containeris made of a transparent material. The inside structure of the containervaries according to the film strength or the arrangement form of thematerial used for the animal cell culture base which will be describedlater. The container is a member partitioned into two parts in order toincorporate necessary members in the inside thereof. After the necessaryinner members (which will be described later) are arranged, all portionsexcept the channels are entirely sealed to prevent liquid leakage. Thecontainer has a supply channel A for supplying a liquid containinganimal cells into the inside thereof, a supply channel B for supplying aculture solution containing nutriments for cultivating animal cells anda substrate solution into the inside thereof, an exhaust channel a forexhausting a liquid containing suspended animal cells from the insidethereof, and an exhaust channel b for exhausting a liquid containing theculture solution and a solution containing a residual part of thesubstrate and the metabolite of animal cells from the inside thereof. Asdescribed above, the container has a series of channels for supplyingand exhausting animal cells and a series of channels for supplying andexhausting both a culture solution and a substrate solution. To supply aliquid into the container without producing bubbles in the liquid, aswill be described later, the supply channel in each channel series isgenerally arranged in a relatively lower portion of the container,whereas the exhaust channel is arranged in the upper portion. Eachchannel may be composed of a hole penetrating the container wall, and aconnection pipe attached thereto. Each connection pipe may be formed ofa cylindrical material, a tubular material or the like selected fromstainless steel, ceramics such as hard glass, etc., heat-resistantpolymer such as polycarbonate, polysulfone, polyethersulfone,polyetheretherketone, etc. and the like. For example, the connectionpipes are respectively connected by means of screws, bolts, or the like,with various kinds of packings interposed therein to prevent liquidleakage in the container wall. Of course, the connection pipes may beformed with the container as a single unit. Opposite ends of the supplychannels are connected to reservoirs for reserving the necessaryliquids.

The animal cell culture base, including a porous carrier and a culturebed applied onto the carrier, is arranged in the inside of the containerso that the liquids supplied from the respective supply channels are ledto the respective exhaust channels after the liquids flow and come incontact therewith. The porous carrier is a structural material forforming the culture bed in a predetermined shape. In respect to thebasic performance of the porous carrier, it is necessary that the porouscarrier be inactive with respect to the culture solution and that thesubstrate solution supplied into the container exhibit a necessaryself-supporting property when immersed in the culture solution and besuitable for application of the culture bed (as will be describedlater). Because the carrier has many pores, it has a very large surface.Further, since there are few dead-end pores, a very large number ofanimal cells with a high density can be grown on the surface of thecarrier. Accordingly, animal cells can be mass-cultivatedsimultaneously, so that the amount of a useful substance produced by theanimal cells in a unit area of the culture base and in a unit time canbe improved greatly.

In the case where the carrier is shaped like a film, the carrier issufficiently flexible that it can be bent or wound to conform to theshape of the container so that it can be placed in the container.Examples of preferred materials for the porous carrier are membrane-likefine porous film, nonwoven fabric, felt, sponge sheet, and the like.Further, the porous carrier is selected from a porous structure in whichhoneycomb-shaped fine vent pores are formed by a porous material,reticular matters are piled up, different-form porous rod-like or linearmatters are used, spherical or different-form porous bead-like or porouspellet-like matters are used as fillers, porous tubular matters are usedas fillers, and porous tubular matters are used in combination, and thelike. The most preferred materials are various kinds of nonwoven fabric.In any case, the nonwoven fabric has a large specific surface area andhas a porous structure constituted by a large number of fine pores.Examples of fiber materials for the nonwoven fabric as the preferredcarrier are polyester fiber, nylon fiber, and polyurethane fiber. Thepreferred material is a fiber material having a fiber packing density ofabout 10 g/m² to about 180 g/m². From the point of view of efficiency inplacing the culture bed in the container, the inside, supply anddiffusion of a culture medium and nutriments for cultivating animalcells in the inside of the nonwoven fabric, and the like, it ispreferable that the thickness of the nonwoven fabric be not larger than5 mm.

The culture bed is applied to the porous carrier. It is preferable thatthe culture bed have a high affinity and a high adhesiveness to theanimal cells and be suitable for adhesion of the animal cells to theculture bed and for cultivating the animal cells with a high yield and ahigh density. The preferred materials for the culture bed are variouskinds of natural or synthetic hydrophilic high-molecular matters havingboth an affinity and an adhesiveness to animal cells. The more preferredare aterrocollagen, poly-vinyl alcohol having a photo cross-linkingradical, polypeptide, and the like. For application of the culture bedonto the porous carrier by using these materials, the carrier isimmersed in an aqueous solution of a matter to form the culture bed,dried by squeezing out excess aqueous solution and then subjected to aninsol-forming treatment. For example, aterrocollagen is made insolubleas follows. Aterrocollagen is applied in the form of an aqueous solutionhaving an adequate aterrocollagen concentration and then treated withammonia and phosphate buffer to be gelated. After being gelated,aterrocollagen is air-dried and then reacted by using ultravioletradiation, glutaraldehyde, hexamethylene disocyanate, sodiumborohydride, epoxy-group cross-linking agent, or the like to introduce across-linking structure to aterrocollagen to thereby make it insoluble.For example, poly-vinyl alcohol having a stilbazolium group as a photocross-linking radical is made insoluble as follows. Poly-vinyl alcoholis applied onto nonwoven fabric by using an aqueous solution containing1-10% by weight of poly-vinyl alcohol and dried at a temperature of 70°C. Then, ultraviolet rays are radiated to the poly-vinyl alcohol for anadequate time to introduce a cross-linking structure to the poly-vinylalcohol to thereby make it insoluble. In particular, the cross-linkedpoly-vinyl alcohol culture medium has a positive (+) surface potential,so that it has a high electrostatic affinity to animal cells having, anegative (-) potential. Further, the culture medium does not inhibit thephysical function of animal cells. Accordingly, the medium is excellentfor adhesion of animal cells. In any case, the medium has an excellentcharacteristic in that it can be used in the form of an aqueous solutionfor application thereof. The matter applied to form the culture bed canbe used if it has a molecular weight suitable for forming a coatingfilm. In most cases, the molecular weight of the applied matter is from10⁵ to 10⁶. The thickness of the culture bed is generally from about 10to about 1000 μm.

The animal cell culture base thus prepared is arranged so that theliquids supplied from the respective supply channels can be led to therespective exhaust channels after the liquids come in contact therewithwhile flowing in the inside of the container. Here, it is important toarrange the culture base so that the supplied liquids can flowefficiently and effectively. In respect to the detailed form of thearrangement, the culture base may be arranged so as to be penetrated bythe liquids supplied into the container or may be arranged so that thesupplied liquids can flow along the culture film. In the case where theanimal cell culture base is shaped like a film, the culture base may bearranged horizontally or may be arranged vertically. In the case wherethe culture film is arrange horizontally, animal cells can becontinuously cultivated without dropping, even if the animal cells areimperfectly adhered in the culture film or are separated from theculture film. Accordingly, in this case, animal cells with a highdensity can be settled on the culture film.

Further, the supply channel A can be divided into groups so thatdifferent liquids can flow to be respectively supplied to spaces betweenculture films in the container. Further, the animal cell culture filmmay be arranged spirally in the container or in the form of a laminateprepared by laminating a large number of sheets or by folding a sheet.In the case where the animal cell culture film is arranged in the formof a laminate or a spiral, supply channels for supplying animal cellsand the culture solution into the container are formed by arrangingseparators between respective culture films and arranged the laminatedculture films at intervals of a short distance. To form gaps suitablefor the dual purpose of supply of animal cells and adhesion thereof tothe culture base, it is preferable that the thickness of each separatoris from about 0.2 to about 2 mm. As a result, the fluidity and diffusionof the liquid containing animal cells supplied from the supply channel Acan be quick and uniform, so that the animal cells can be distributedand uniformly to the entire surface of the culture bed. Furthermore,there is another effect in that the culture solution penetrating theculture films can be further diffused and mixed in the gaps formed bythe presence of the separators to thereby attain uniformity ofcomponents. The respective separator may be formed of a material havingrough gaps or pores. Examples of the preferred form are the form of aporous film or net having a rough pore diameter.

As will be described later by way of example, a rough porous supportingplate may be used for supporting the opposite ends of the culture baseand the separator. The supporting plate functions to support theopposite ends of the culture base and the separator and to supply theanimal cells from the entire surface of the supporting plate to thecontainer. Accordingly, it is necessary that the supporting plate havecontinuous through pores with a pore diameter of about 50 to 200 μm toavoid abrasion of the supplied animal cells caused by the flowingthereof. The preferred material for the porous supporting plate is ahard material or a relatively highly elastic material.

As will be described later, a microfiltration membrane is arrangedacross the exhaust channel b. Examples of the form of themicrofiltration membrane are a flat film, a pleat, a tube, a hollowfiber, a fine porous tube, and the like. The microfiltration membraneprevents the animal cells and the medium suspended from the first orsuspended by dropping out of the culture base after adsorbing theretofrom flowing out of the container and, at the same time, filtrates auseful substrate produced by the animal cells, to thereby continuouslyexhaust it to the outside of the container. Since the pressure of theexhausted liquid acts on the microfiltration membrane 6 arranged acrossthe exhaust channel b as described above, sufficient consideration mustbe given on the settlement of the microfiltration membrane to preventthe membrane from being dislodged in the liquid-feeding operation. Aswill be described later in the first embodiment, the microfiltrationmembrane 6 is secured between side walls 12 and then the upper and lowermembers 13, 14 as illustrated in FIG. 9. At the same time, themicrofiltration membrane 6 is placed into the container 1 by compressingor by forcedly inserting the membrane in a frame.

The microfiltration membrane is made of a material having a durabilityagainst vapor sterilizing treatment. Examples of the materials for themicrofiltration membrane are porous films such as a flat film, a pleatfilm, a tubular film, a hollow fiber film, a fine tubular film, etc.,having a pore diameter of about 1 μm to the order of tens of microns andbeing selected from polysulfone, polyethersulfone, poly (vinylidenefluoride), nylon, etc. It is preferable that the material doesn't adhereprotein, doesn't occur fouling and doesn't become blocked. Examples ofavailable materials for the microfiltration membrane are "PSE-20","PSE-45", "PSE-80", "PSE-200" (all tradenames produced by Fuji PhotoFilm Co., Ltd.), etc. These available materials can be used in theapparatus of the present invention.

In the case where the animal cell culture base is shaped like a film andarranged in the form of a spiral as described above, the container canbe provided in the form of a cylindrical container capable of beingrotated to provide an apparatus improved in fluidity, diffusion anduniformity of various kinds of liquids supplied into the container.

By using the bioreactor apparatus according to the present invention asconstructed above, animal cells are cultivated as follows. The inside ofthe container is subjected to a sterilizing or germicidal treatment byheating the container or by introducing high-temperature vapor, hotwater, aqueous alcohol having an adequate concentration or germicide gassuch as ethylene oxide gas, etc. into the container so as to becirculated, if necessary, and then radiated with radiating rays such asultraviolet rays, λ-rays, etc. In the case where the container or themedium of the treatment is heated in this treatment, it is heated at atemperature of about 120 to about 130° C. for a period of about 5 toabout 30 minutes.

After the container sterilizing treatment is finished, the supply pipeof the supply channel A is connected to a tank reserving the liquidcontaining animal cells whereas the exhaust pipe of the exhaust channela is connected to a reservoir or is connected to the aforementioned tankif the liquid is circulated. The supply pipe of the supply channel B isconnected to a tank reversing the culture solution and the substratesolution whereas the exhaust pipe of the exhaust channel b is connectedto an exhaust liquid reservoir.

Then, a liquid containing animal cells is supplied from the supplychannel A. The container is filled with the supplied liquid, so that theliquid penetrates the animal cell culture base arranged in the inside ofthe container. As a result, animal cells are adhered in the culture bedof the culture base. To improve the density of animal cells adhered tothe culture bed, the liquid may be introduced into the container againfrom the supply channel A so as to be circulated between the containerand the tank.

The kind of animal cells used in the apparatus of the invention is notlimited specifically. Examples of the animal cells are T cell, B cell,killer cell, human tumor cell, fibroblast cell, lymphoblast cell, EBvirus mutant cell, etc. These components are respectively mixed anddiffused to a concentration ranging approximately from 1 to 100 mg/l(animal cells) with respect to a solvent, such as water. Various kindsof amino acids, vitamins and various kinds of saccharides as anotherculture medium may be added to the liquid containing animal cells.

Immediately after the supply of animal cells to the culture base isfinished, an animal cell culture solution and a substrate solution aresupplied from the supply pipe of the supply channel B. These culture andsubstrate solutions are diffused or fluidized into the container andbrought into contact with animal cells on the culture base to therebycontribute to the growth or culture thereof.

The culture solution contains, as main components, nutriments necessaryfor the growth of animal cells. Examples of the nutriments are variouskinds of essential amino acids, various kinds of vitamins, saccharidessuch as glucose, serum components, etc. These nutriments are provided inthe form of an aqueous solution having a concentration of about 1 toabout 100 g/l. Because the substrate serves as a raw material forproducing a necessary metabolite, a specific metabolite can beselectively produced by specifying the kind of the animal cells andsubstrate. Examples of the substrate are essential amino acids, variouskinds of vitamins, saccharides such as glucose, serum components, etc.The components of the culture solution may be physically equal to thoseof the substrate but the former is different from the latter in that theformer is supplied at the time of cultivating animal cells and for thepurpose of cultivating animal cells at the time of cultivating, whereasthe latter is supplied for the cultivated or grown animal cells toproduce metabolites thereof. The substrate solution is prepared in theform of an aqueous solution having a concentration of about 1 to about100 g/l, with respect to the aforementioned components.

The supply flow rate in each of the liquid containing animal cells, theculture solution and the substrate solution ranges from about 1 to about100 ml per minute. The supply flow rate is suitably adjusted accordingto the adhesive ability of animal cells to the culture bed, the growthrate thereof, the degree of the growth thereof, and the like. Theseliquids can be recycled between the container and the tank if they canbe repeatedly used with no trouble.

For the growth of animal cells, it is preferable that dissolved oxygenbe contained in the supplied liquid. It is most preferable that oxygenbe saturated. The saturated oxygen concentration is generally about 10mg/l. Such dissolved oxygen may be contained in any liquid supplied intothe container. Further, each of the liquids may contain other necessarycomponents.

The culture solution is continuously supplied to animal cells adhered inthe culture bed while factors such as osmotic pressure, dissolved oxygenvolume, pH, liquid temperature, substrate concentration of the substratesolution, metabolite concentration, etc. are continuously controlled toan optimum range suitable for cultivating animal cells, by which animalcells can be cultivated with a high yield and a high density.

The animal cells cultivated by the aforementioned operation produce ametabolite by reaction with the substrate. The metabolite is exhaustedfrom the exhaust channel b through the microfiltration membrane, asdescribed above, and collected. Then, the metabolite is extracted fromthe exhaust liquid and purified to a useful substance. The metabolitevaries according to the kind of the animal cells and the substrate giventhereto. Examples of the metabolite are cure vaccines, interferons,monoclonal antibodies, cancer antigens, hormones, cell growth factors,lymphocines, various kinds of catalysts and the like.

The present invention will be described more in detail as to thepreferred embodiments illustrated in the drawings in which like numeralsrepresent like parts. FIG. 1 is a sectional view showing the schematicconfiguration of a bioreactor using a stratified animal cell culturefilm as an embodiment of the present invention. In the drawing,container 1 is composed of side walls 11 and 12, a lower wall 13, and anupper wall 14 (and front and back walls, not shown). The container 1 isformed by combination of the respective walls as illustrated in thedrawings. Liquid channels composed of through holes 111, 121, 131 and141 for penetrating the walls and connection pipes 21, 22, 23 and 24 areformed at predetermined positions of the respective walls, respectively.In this embodiment, the reference numeral 23 designates a supply channelA for supplying a liquid containing animal cells into the container 1,24 an exhaust channel a for exhausting the liquid supplied from thechannel 23 to discharge it or recycle it to the apparatus, 21 a supplychannel B for supplying a liquid containing a culture solutioncontaining nutriments to be given to animal cells supported in thecontainer and 22 an exhaust channel b for exhausting the liquid suppliedfrom the supply channel 21 out of the container 1 to discharge it orrecycle it from the channel 22 to the apparatus and for exhausting aliquid containing a metabolite produced by the animal cells out of thecontainer 1 to collect the metabolite.

Cavities 112 and 122 are respectively provided in the inside of the wallmembers 11 and 12 of the container 11. The cavity 112 serves as a liquidreservoir for the liquid supplied from the channel 21 to increase theflow rate of the liquid in the supply channel B. The cavity 122 servesas a liquid reservoir for the liquid exhausted from the channel 22 tolead the flow of the liquid to the exhaust channel b. Accordingly, thecavities can increase the liquid flow efficiency of the respectiveliquids. In the apparatus as shown in the drawing, the shell of thecontainer 1 is formed by connecting the lower and upper walls 13 and 14to the upper and lower portions of the side walls 11 and 12 (and thefront and back walls) by means of riveting or screwing. In the inside ofthe container, a rough porous supporting plate 33 contacts the innersurface of the lower wall 13, a rough porous supporting plate 34contacts the inner surface of the upper wall 14 and a rough poroussupporting plate 31 contacts the inner surface of the side wall 11.

As described above, animal cell culture films 4 and sheet-likeseparators 5 are successively alternately laminated and packed in aspace surrounded by the rough porous plates 31, 33 and 34. Amicrofiltration membrane 6 is arranged between the outermost layer ofthe laminate and the wall member 12 of the container. Because aconsiderably large amount of pressure caused by the small size of thepore diameter and the blocking of the pores in the cultivating periodacts on the microfiltration membrane 6, it is necessary to compress themicrofiltration membrane 6 between the container wall members 12 and 13and between the wall members 12 and 14, as shown in the drawing.

After the members are arranged in the container as described above andthe container wall members are combined with each other, a sterilizingtreatment is applied by passing high-temperature vapor from the supplychannel A to the exhaust channel b and from the supply channel B to theexhaust channel a for about 30 minutes. Then, an aqueous solutioncontaining about 1 g/l of specific animal cells is supplied from thesupply channel A to the container 1 to produce a predeterminedmetabolite. The cell-containing solution enters into the container,reaches the rough porous plate 34 so as to be diffused to the entiresurface thereof, propagates between the separators 5 in the container 1to fill the container and at the same time penetrates the pore tissuesof the culture films 4. The animal cells in the aqueous solution sinkonto the tissue surface of the culture films and are then adheredthereto. With the supply of the cell-containing solution, the amount ofthe adhered cells is increased. The time required for the adhesion of asufficient amount of the animal cells is from about 30 to about 60minutes.

After the adhesion of the sufficient amount of the animal cells isconfirmed, the supply of the animal cell culture solution from thesupply channel B is started. The flow rate of the supplied culturesolution is about 10 liters per day.

With the supply of the culture solution to the container 1, the animalcell-containing solution in the container 1 is gradually replaced by theculture solution. The entire replacement is finished about 30 minutesafter the starting of the supply. Then, the supply of the culturesolution is continued for about 24 hours, so that the animal cells inthe container 1 are uniformly grown. As a result, animal cells can becultivated to a high yield and a high density in the bioreactorapparatus of the invention. Therefore, a predetermined substrate issupplied for a period of one day to 3 days to produce a predeterminedyield of the metabolite.

FIG. 2 is a sectional view showing the schematic configuration of abioreactor using a spiral-shaped animal cell culture film, as anotherembodiment of the present invention. In the drawing, the container 1,which is different from that in the embodiment shown in FIG. 1, iscomposed of a tubular wall member 10, a lower wall member 13, and anupper wall member 14. This embodiment is similar to the previousembodiment (FIG. 1) in that two channel series (the supply channel A(23), the exhaust channel a (24), the supply channel B (21) and theexhaust channel b (22)) are provided in the container 1. In theembodiment shown in FIG. 2, the exhaust channel b (22), which isprovided in the side wall member in the previous embodiment, is providedin the upper wall member. A hollow tube 7 having a large number of poresat the sides thereof is arranged at the center of the inside of thecontainer. The upper end of the hollow tube 7 is connected to the centerportion of the upper wall member 14 at a place containing the exhaustchannel b in the inside thereof. That is, the hollow tube 7 is suspendedfrom the upper wall member 14. A microfiltration membrane 6 is wound onthe outer circumference of the hollow tube 7 so that the hollow tube 7is coated with the microfiltration membrane 6. A culture film 4 and asheet-like separator 5 are alternately wound on the outer circumferenceof the microfiltration membrane 6. The elements are packed in thecontainer 1 and fixed in the inside of the container 1 by settingO-rings 8 to the upper and lower ends thereof on the outer circumferencethereof. The gap between the side tubular wall 10 formed by the O-rings8 and the wound matter is a space having the same function as that ofthe cavities 112 and 122 in the wall member in the previous embodiment.A rough porous plate 33 is arranged on the lower wall member 13 in thesame manner as in the previous embodiment. A rough porous plate 34 isalso arranged to the inner surface of the upper wall member 14. Theopposite ends of the wound matter are fixed closely and tightly in thecontainer 1 by the two porous plates.

A sterilizing treatment is applied to the apparatus of the invention bypassing high-temperature vapor from the supply channel A to the exhaustchannel b and from the supply channel B to the exhaust channel a forabout 30 minute. Then, an aqueous solution containing about 1 g/l ofspecific animal cells is supplied from the supply channel A to thecontainer 1 to produce a predetermined metabolite. The cell-containingsolution enters into the container 1, reaches the rough porous plate 34so as to be diffused to the whole surface thereof, propagates betweenthe separators 5 in the container 1 to fill the container 1 and at thesame time penetrates the pore tissue of the culture films 4. The animalcells in the aqueous solution contact onto the tissue surface of theculture films and then become adhered thereto. With the supply of thecell-containing solution, the amount of the adhered cells is increased.The time required for the adhesion of the sufficient amount of theanimal cells is from about 30 to about 60 minutes.

After the deposition of the sufficient amount of the animal cells isconfirmed, the supply of the animal cell-containing solution from thesupply channel A is terminated and the supply of the culture solutionfrom the supply channel B is started. The flow rate of the suppliedculture solution is about 10 liters per day.

With the supply of the culture solution to the container 1, the animalcell-containing solution in the container 1 is gradually replaced by theculture solution. The entire replacement is finished about 30 minutesafter the starting of the supply. Then, the supply of the culture iscontinued for about 24 hours, so that the animal cells in the container1 are uniformly grown. As a result, animal cells can be cultivated to ahigh yield and a high density in the bioreactor apparatus of theinvention. Then, a predetermined substrate is supplied for a period ofone day to 3 days to produce a predetermined yield of the metabolite.

According to the bioreactor apparatus of the invention, animal cellswith a high yield and a high density are deposited on the culture basearranged in the inside of the container and cultivated by liquidcirculation culture technique. Accordingly, the supply and exchange ofthe culture solution can be performed automatically with ease. Further,an efficient metabolic reaction of animal cells can be performed bycontinuously controlling factors such as osmotic pressure, dissolvedcatalyst, pH, temperature, substrate concentration and metaboliteconcentration to optimum ranges suitable for cultivating the cells.Further, because the animal cells, the culture solution, the substrateand other necessary elements can be supplied in parallel to the culturebed carrier or perpendicularly thereto, these elements can be broughtinto efficient contact with the animal cells. Because the animal cellsare deposited or fixed to the carrier so that there is no contactbetween the animal cells and between parts of the carrier, shear stressimposed on the cells is so small that inactivation of the cellsinfrequently occurs. Further, because the produced metabolite iscontinuously took out of the apparatus through the microfiltrationmembrane, there is no interference of the animal cells with themetabolite. As a result, a metabolic reaction of the animal cellsprogresses efficiently in the container.

What is claimed is:
 1. A bioreactor apparatus, comprising:a hermeticallysealed container having a first inlet disposed on a first wall forsupplying a culture broth containing suspended animal cells to seedanimal cells into the inside of said container; a second inlet forsupplying and circulating a culture solution or a substrate solutioninto the inside of said container; a first outlet disposed on a secondwall opposite said first wall for exhausting gas and an excess part ofthe culture broth from the inside of said container; a second outlet forexhausting and circulating a residual part of the culture solution orthe substrate solution and a solution containing metabolites of theanimal cells from the inside of said container; a first porous supportplate disposed on an inner surface of said first wall so as to coversaid first inlet; a second porous support plate disposed on an innersurface of said second wall so as to cover said first outlet; an animalcell culture base comprising at least one porous sheet carrier havingsides coated with a culture bed layer suitable for adhesion of theanimal cells, said at least one porous sheet carrier being positionedwithin the container such that a plurality of flow channels are formedbetween said sides, said channels being formed by spacers positionedbetween said sides and said channels extending between said first poroussupport plate and said second porous support plate such that saidculture broth supplied from said first inlet flows through said firstporous support plate to each of said channels and flows out of saidchannels through said second porous support plate to said first outlet;and a microfiltration membrane positioned within said container so as tocover said second outlet, said membrane for retaining suspended animalcells inside said container while allowing the passage of metabolitesproduced by the animal cells, said second inlet and said second outletbeing positioned such that said culture solution or substrate solutionpenetrates the sides of said at least one sheet carrier while flowingfrom said second inlet through said membrane and out of said secondoutlet.
 2. The apparatus of claim 1, wherein said at least one poroussheet carrier is made of nonwoven fabric.
 3. The apparatus of claim 1,wherein said at least one porous sheet carrier is planar.
 4. Theapparatus of claim 1, wherein said culture base comprises a plurality ofsaid porous sheet carriers wherein each of said sheet carriers is spacedfrom the other by said spacers so as to form said plurality of flowchannels.
 5. A bioreactor apparatus, comprising:a hermetically sealedtubular container including a tubular wall, a first sealed end and asecond sealed end; a first inlet disposed on said first sealed end forsupplying a culture broth containing suspended animal cells to seedanimal cells into the inside of said container; a second inlet forsupplying and circulating a culture solution or a substrate solutioninto the inside of said container; a first outlet disposed on saidsecond sealed end for exhausting gas and an excess part of the culturebroth from the inside of said container; a second outlet for exhaustingand circulating a residual part of the culture solution or the substratesolution and a solution containing metabolites of the animal cells fromthe inside of said container; a first porous support plate disposed onan inner surface of said first sealed end so as to cover said firstinlet; a second porous support plate disposed on an inner surface ofsaid second sealed end so as to cover said first inlet; a hollow tubeextending axially in said container so as to define an annular spacetherebetween, said tube having pores on the outer circumference thereofand said second outlet being in flow communication with the inside ofsaid tube; an animal cell culture base comprising at least one poroussheet carrier having sides coated with a culture bed layer suitable foradhesion of the animal cells, said at least one porous sheet carrierbeing spirally wound within said annular space such that a plurality offlow channels are formed between said sides, said channels being formedby spacers positioned between said sides and said channels extendingbetween said first porous support plate and said second porous supportplate such that said culture broth supplied from said first inlet flowsthrough said first porous support plate to each of said channels andflows out of said channels through said second porous support plate tosaid first outlet; and a microfiltration membrane circumscribing saidtube in the inside of said container, said membrane for retainingsuspended animal cells inside said container while allowing the passageof metabolites produced by the animal cells, said second inlet beingpositioned such that said culture solution or substrate solutionsupplied by said second inlet penetrates the sides of said at least onesheet carrier while radially flowing from said second inlet through saidmembrane and out of said second outlet.
 6. The apparatus of claim 5,wherein said second inlet is disposed on said tubular wall.
 7. Theapparatus of claim 6, wherein said second outlet is disposed on the sameend as said first outlet.